Human kinases and polynucleotides encoding the same

ABSTRACT

Novel human polynucleotide and polypeptide sequences are disclosed that can be used in therapeutic, diagnostic, and pharmacogenomic applications.

The present application claims the benefit of U.S. ProvisionalApplication No. 60/239,821, which was filed on Oct. 12, 2000, and isherein incorporated by reference in its entirety.

1. INTRODUCTION

The present invention relates to the discovery, identification, andcharacterization of novel human polynucleotides encoding proteinssharing sequence similarity with animal kinases. The inventionencompasses the described polynucleotides, host cell expression systems,the encoded proteins, fusion proteins, polypeptides and peptides,antibodies to the encoded proteins and peptides, and geneticallyengineered animals that either lack or over express the disclosed genes,antagonists and agonists of the proteins, and other compounds thatmodulate the expression or activity of the proteins encoded by thedisclosed genes that can be used for diagnosis, drug screening, clinicaltrial monitoring, the treatment of diseases and disorders, and cosmeticor nutriceutical applications.

2. BACKGROUND OF THE INVENTION

Kinases mediate the phosphorylation of a wide variety of proteins andcompounds in the cell. Along with phosphatases, kinases are involved ina range of regulatory pathways. Given the physiological importance ofkinases, they have been subject to intense scrutiny and are proven drugtargets.

3. SUMMARY OF THE INVENTION

The present invention relates to the discovery, identification, andcharacterization of nucleotides that encode novel human proteins and thecorresponding amino acid sequences of these proteins. The novel humanproteins (NHPs) described for the first time herein share structuralsimilarity with animal kinases, including, but not limited to,serine-threonine kinases, calcium/calmodulin-dependent protein kinases,and mitogen activated kinases. Accordingly, the described NHPs encodenovel kinases having homologues and orthologs across a range of phylaand species.

The novel human polynucleotides described herein, encode open readingframes (ORFs) encoding proteins of 766 and 765 amino acids in length(see respectively SEQ ID NOS: 2 and 4).

The invention also encompasses agonists and antagonists of the describedNHPs, including small molecules, large molecules, mutant NHPs, orportions thereof, that compete with native NHP, peptides, andantibodies, as well as nucleotide sequences that can be used to inhibitthe expression of the described NHPs (e.g., antisense and ribozymemolecules, and open reading frame or regulatory sequence replacementconstructs) or to enhance the expression of the described NHPs (e.g.,expression constructs that place the described polynucleotide under thecontrol of a strong promoter system), and transgenic animals thatexpress a NHP sequence, or “knock-outs” (which can be conditional) thatdo not express a functional NHP. Knock-out mice can be produced inseveral ways, one of which involves the use of mouse embryonic stemcells (“ES cells”) lines that contain gene trap mutations in a murinehomolog of at least one of the described NHPs. When the unique NHPsequences described in SEQ ID NOS:1-4 are “knocked-out” they provide amethod of identifying phenotypic expression of the particular gene aswell as a method of assigning function to previously unknown genes. Inaddition, animals in which the unique NHP sequences described in SEQ IDNOS:1-4 are “knocked-out” provide a unique source in which to elicitantibodies to homologous and orthologous proteins that would have beenpreviously viewed by the immune system as “self” and therefore wouldhave failed to elicit significant antibody responses. To these ends,gene trapped knockout ES cells have been generated in murine homologs ofthe described NHPs.

Additionally, the unique NHP sequences described in SEQ ID NOS:1-4 areuseful for the identification of protein coding sequence and mapping aunique gene to a particular chromosome. These sequences identifybiologically verified exon splice junctions as opposed to splicejunctions that may have been bioinformatically predicted from genomicsequence alone. The sequences of the present invention are also usefulas additional DNA markers for restriction fragment length polymorphism(RFLP) analysis, and in forensic biology.

Further, the present invention also relates to processes for identifyingcompounds that modulate, i.e., act as agonists or antagonists, of NHPexpression and/or NHP activity that utilize purified preparations of thedescribed NHPs and/or NHP product, or cells expressing the same. Suchcompounds can be used as therapeutic agents for the treatment of any ofa wide variety of symptoms associated with biological disorders orimbalances.

4. DESCRIPTION OF THE SEQUENCE LISTING AND FIGURES

The Sequence Listing provides the sequence of the novel human ORFsencoding the described novel human kinase proteins.

5. DETAILED DESCRIPTION OF THE INVENTION

The NHPs described for the first time herein are novel proteins that areexpressed in, inter alia, human cell lines and human fetal brain, brain,pituitary, spinal cord, testis, adipose, and esophagus cells. Thedescribed sequences were compiled from sequences available in GENBANK,and cDNAs generated from skeletal muscle, adipose, pituitary,cerebellum, and brain mRNA (Edge Biosystems, Gaithersburg, Md.).

The present invention encompasses the nucleotides presented in theSequence Listing, host cells expressing such nucleotides, the expressionproducts of such nucleotides, and: (a) nucleotides that encode mammalianhomologs of the described genes, including the specifically describedNHPS, and the NHP products; (b) nucleotides that encode one or moreportions of an NHP that correspond to functional domains, and thepolypeptide products specified by such nucleotide sequences, includingbut not limited to the novel regions of any active domain(s); (c)isolated nucleotides that encode mutant versions, engineered ornaturally occurring, of the described NHPs in which all or a part of atleast one domain is deleted or altered, and the polypeptide productsspecified by such nucleotide sequences, including but not limited tosoluble proteins and peptides in which all or a portion of the signalsequence is deleted; (d) nucleotides that encode chimeric fusionproteins containing all or a portion of a coding region of a NHP, or oneof its domains (e.g., a receptor/ligand binding domain, accessoryprotein/self-association domain, etc.) fused to another peptide orpolypeptide; or (e) therapeutic or diagnostic derivatives of thedescribed polynucleotides such as oligonucleotides, antisensepolynucleotides, ribozymes, dsRNA, or gene therapy constructs comprisinga sequence first disclosed in the Sequence Listing. As discussed above,the present invention includes: (a) the human DNA sequences presented inthe Sequence Listing (and vectors comprising the same) and additionallycontemplates any nucleotide sequence encoding a contiguous NHP openreading frame (ORF) that hybridizes to a complement of a DNA sequencepresented in the Sequence Listing under highly stringent conditions,e.g., hybridization to filter-bound DNA in 0.5 M NaHPO₄, 7% sodiumdodecyl sulfate (SDS), 1 mM EDTA at 65° C., and washing in 0.1×SSC/0.1%SDS at 68° C. (Ausubel et al., eds., 1989, Current Protocols inMolecular Biology, Vol. I, Green Publishing Associates, Inc., and JohnWiley & sons, Inc., New York, at p. 2.10.3) and encodes a functionallyequivalent expression product. Additionally, contemplated are anynucleotide sequences that hybridize to the complement of the DNAsequence that encode and express an amino acid sequence presented in theSequence Listing under moderately stringent conditions, e.g., washing in0.2×SSC/0.1% SDS at 42° C. (Ausubel et al., 1989, supra), yet stillencode a functionally equivalent NHP product. Functional equivalents ofa NHP include naturally occurring NHPs present in other species andmutant NHPs whether naturally occurring or engineered (by site directedmutagenesis, gene shuffling, directed evolution as described in, forexample, U.S. Pat. Nos. 5,837,458 or 5,723,323, both of which are hereinincorporated by reference). The invention also includes degeneratenucleic acid variants of the disclosed NHP polynucleotide sequences.

Additionally contemplated are polynucleotides encoding NHP ORFs, ortheir functional equivalents, encoded by polynucleotide sequences thatare about 99, 95, 90, or about 85 percent similar to correspondingregions of SEQ ID NO:1 (as measured by BLAST sequence comparisonanalysis using, for example, the GCG sequence analysis package usingdefault parameters).

The invention also includes nucleic acid molecules, preferably DNAmolecules, that hybridize to, and are therefore the complements of, thedescribed NHP encoding polynucleotides. Such hybridization conditionscan be highly stringent or less highly stringent, as described above. Ininstances where the nucleic acid molecules are deoxyoligonucleotides(“DNA oligos”), such molecules are generally about 16 to about 100 baseslong, or about 20 to about 80, or about 34 to about 45 bases long, orany variation or combination of sizes represented therein thatincorporate a contiguous region of sequence first disclosed in theSequence Listing. Such oligonucleotides can be used in conjunction withthe polymerase chain reaction (PCR) to screen libraries, isolate clones,and prepare cloning and sequencing templates, etc.

Alternatively, such NHP oligonucleotides can be used as hybridizationprobes for screening libraries, and assessing gene expression patterns(particularly using a micro array or high-throughput “chip” format).Additionally, a series of the described NHP oligonucleotide sequences,or the complements thereof, can be used to represent all or a portion ofthe described NHP sequences. An oligonucleotide or polynucleotidesequence first disclosed in at least a portion of one or more of thesequences of SEQ ID NOS: 1-4 can be used as a hybridization probe inconjunction with a solid support matrix/substrate (resins, beads,membranes, plastics, polymers, metal or metallized substrates,crystalline or polycrystalline substrates, etc.) . Of particular noteare spatially addressable arrays (i.e., gene chips, microtiter plates,etc.) of oligonucleotides and polynucleotides, or correspondingoligopeptides and polypeptides, wherein at least one of the biopolymerspresent on the spatially addressable array comprises an oligonucleotideor polynucleotide sequence first disclosed in at least one of thesequences of SEQ ID NOS: 1-4, or an amino acid sequence encoded thereby.Methods for attaching biopolymers to, or synthesizing biopolymers on,solid support matrices, and conducting binding studies thereon aredisclosed in, inter alia, U.S. Pat. Nos. 5,700,637, 5,556,752,5,744,305, 4,631,211, 5,445,934, 5,252,743, 4,713,326, 5,424,186, and4,689,405 the disclosures of which are herein incorporated by referencein their entirety.

Addressable arrays comprising sequences first disclosed in SEQ IDNOS:1-4 can be used to identify and characterize the temporal and tissuespecific expression of a gene. These addressable arrays incorporateoligonucleotide sequences of sufficient length to confer the requiredspecificity, yet be within the limitations of the production technology.The length of these probes is within a range of between about 8 to about2000 nucleotides. Preferably the probes consist of 60 nucleotides andmore preferably 25 nucleotides from the sequences first disclosed in SEQID NOS:1-4.

For example, a series of the described oligonucleotide sequences, or thecomplements thereof, can be used in chip format to represent all or aportion of the described sequences. The oligonucleotides, typicallybetween about 16 to about 40 (or any whole number within the statedrange) nucleotides in length can partially overlap each other and/or thesequence may be represented using oligonucleotides that do not overlap.Accordingly, the described polynucleotide sequences shall typicallycomprise at least about two or three distinct oligonucleotide sequencesof at least about 8 nucleotides in length that are each first disclosedin the described Sequence Listing. Such oligonucleotide sequences canbegin at any nucleotide present within a sequence in the SequenceListing and proceed in either a sense (5′-to-3′) orientation vis-a-visthe described sequence or in an antisense orientation.

Microarray-based analysis allows the discovery of broad patterns ofgenetic activity, providing new understanding of gene functions andgenerating novel and unexpected insight into transcriptional processesand biological mechanisms. The use of addressable arrays comprisingsequences first disclosed in SEQ ID NOS:1-4 provides detailedinformation about transcriptional changes involved in a specificpathway, potentially leading to the identification of novel componentsor gene functions that manifest themselves as novel phenotypes.

Probes consisting of sequences first disclosed in SEQ ID NOS:1-4 canalso be used in the identification, selection and validation of novelmolecular targets for drug discovery. The use of these unique sequencespermits the direct confirmation of drug targets and recognition of drugdependent changes in gene expression that are modulated through pathwaysdistinct from the drugs intended target. These unique sequencestherefore also have utility in defining and monitoring both drug actionand toxicity.

As an example of utility, the sequences first disclosed in SEQ IDNOS:1-4 can be utilized in microarrays or other assay formats, to screencollections of genetic material from patients who have a particularmedical condition. These investigations can also be carried out usingthe sequences first disclosed in SEQ ID NOS:1-4 in silico and bycomparing previously collected genetic databases and the disclosedsequences using computer software known to those in the art.

Thus the sequences first disclosed in SEQ ID NOS:1-4 can be used toidentify mutations associated with a particular disease and also as adiagnostic or prognostic assay.

Although the presently described sequences have been specificallydescribed using nucleotide sequence, it should be appreciated that eachof the sequences can uniquely be described using any of a wide varietyof additional structural attributes, or combinations thereof. Forexample, a given sequence can be described by the net composition of thenucleotides present within a given region of the sequence in conjunctionwith the presence of one or more specific oligonucleotide sequence(s)first disclosed in the SEQ ID NOS: 1-4. Alternatively, a restriction mapspecifying the relative positions of restriction endonuclease digestionsites, or various palindromic or other specific oligonucleotidesequences can be used to structurally describe a given sequence. Suchrestriction maps, which are typically generated by widely availablecomputer programs (e.g., the University of Wisconsin GCG sequenceanalysis package, SEQUENCHER 3.0, Gene Codes Corp., Ann Arbor, Mich.,etc.), can optionally be used in conjunction with one or more discretenucleotide sequence(s) present in the sequence that can be described bythe relative position of the sequence relative to one or more additionalsequence(s) or one or more restriction sites present in the disclosedsequence.

For oligonucleotide probes, highly stringent conditions may refer, e.g.,to washing in 6×SSC/0.05% sodium pyrophosphate at 37° C. (for 14-baseoligos), 48° C. (for 17-base oligos), 55° C. (for 20-base oligos), and60° C. (for 23-base oligos). These nucleic acid molecules may encode oract as NHP gene antisense molecules, useful, for example, in NHP generegulation (for and/or as antisense primers in amplification reactionsof NHP gene nucleic acid sequences). With respect to NHP generegulation, such techniques can be used to regulate biologicalfunctions. Further, such sequences can be used as part of ribozymeand/or triple helix sequences that are also useful for NHP generegulation.

Inhibitory antisense or double stranded oligonucleotides canadditionally comprise at least one modified base moiety that is selectedfrom the group including but not limited to 5-fluorouracil,5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine,4-acetylcytosine, 5-(carboxyhydroxylmethyl) uracil,5-carboxymethylaminomethyl-2-thiouridine,5-carboxymethylaminomethyluracil, dihydrouracil,beta-D-galactosylqueosine, inosine, N6-isopentenyladenine,1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine,2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine,7-methylguanine, 5-methylaminomethyluracil,5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine,5′-methoxycarboxymethyluracil, 5-methoxyuracil,2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid (v),wybutoxosine, pseudouracil, queosine, 2-thiocytosine,5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil,uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid (v),5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl) uracil, (acp3)w,and 2,6-diaminopurine.

The antisense oligonucleotide can also comprise at least one modifiedsugar moiety selected from the group including but not limited toarabinose, 2-fluoroarabinose, xylulose, and hexose.

In yet another embodiment, the antisense oligonucleotide will compriseat least one modified phosphate backbone selected from the groupconsisting of a phosphorothioate, a phosphorodithioate, aphosphoramidothioate, a phosphoramidate, a phosphordiamidate, amethylphosphonate, an alkyl phosphotriester, and a formacetal or analogthereof.

In yet another embodiment, the antisense oligonucleotide is anα-anomeric oligonucleotide. An α-anomeric oligonucleotide forms specificdouble-stranded hybrids with complementary RNA in which, contrary to theusual β-units, the strands run parallel to each other (Gautier et al.,1987, Nucl. Acids Res. 15:6625-6641). The oligonucleotide is a2′-0-methylribonucleotide (Inoue et al., 1987, Nucl. Acids Res.15:6131-6148), or a chimeric RNA-DNA analogue (Inoue et al., 1987, FEBSLett. 215:327-330). Alternatively, double stranded RNA can be used todisrupt the expression and function of a targeted NHP.

Oligonucleotides of the invention can be synthesized by standard methodsknown in the art, e.g., by use of an automated DNA synthesizer (such asare commercially available from Biosearch, Applied Biosystems, etc.). Asexamples, phosphorothioate oligonucleotides can be synthesized by themethod of Stein et al. (1988, Nucl. Acids Res. 16:3209), andmethylphosphonate oligonucleotides can be prepared by use of controlledpore glass polymer supports (Sarin et al., 1988, Proc. Natl. Acad. Sci.U.S.A. 85:7448-7451), etc.

Low stringency conditions are well known to those of skill in the art,and will vary predictably depending on the specific organisms from whichthe library and the labeled sequences are derived. For guidanceregarding such conditions see, for example, Sambrook et al., 1989,Molecular Cloning, A Laboratory Manual (and periodic updates thereof),Cold Springs Harbor Press, N.Y.; and Ausubel et al., 1989, supra.

Alternatively, suitably labeled NHP nucleotide probes can be used toscreen a human genomic library using appropriately stringent conditionsor by PCR. The identification and characterization of human genomicclones is helpful for identifying polymorphisms (including, but notlimited to, nucleotide repeats, microsatellite alleles, singlenucleotide polymorphisms, or coding single nucleotide polymorphisms),determining the genomic structure of a given locus/allele, and designingdiagnostic tests. For example, sequences derived from regions adjacentto the intron/exon boundaries of the human gene can be used to designprimers for use in amplification assays to detect mutations within theexons, introns, splice sites (e.g., splice acceptor and/or donor sites),etc., that can be used in diagnostics and pharmacogenomics.

For example, the present sequences can be used in restriction fragmentlength polymorphism (RFLP) analysis to identify specific individuals. Inthis technique, an individual's genomic DNA is digested with one or morerestriction enzymes, and probed on a Southern blot to yield unique bandsfor identification (as generally described in U.S. Pat. No. 5,272,057,incorporated herein by reference). In addition, the sequences of thepresent invention can be used to provide polynucleotide reagents, e.g.,PCR primers, targeted to specific loci in the human genome, which canenhance the reliability of DNA-based forensic identifications by, forexample, providing another “identification marker” (i.e., another DNAsequence that is unique to a particular individual). Actual basesequence information can be used for identification as an accuratealternative to patterns formed by restriction enzyme generatedfragments.

Further, a NHP gene homolog can be isolated from nucleic acid from anorganism of interest by performing PCR using two degenerate or “wobble”oligonucleotide primer pools designed on the basis of amino acidsequences within the NHP products disclosed herein. The template for thereaction may be total RNA, mRNA, and/or cDNA obtained by reversetranscription of mRNA prepared from, for example, human or non-humancell lines or tissue known or suspected to express an allele of a NHPgene.

The PCR product can be subcloned and sequenced to ensure that theamplified sequences represent the sequence of the desired NHP gene. ThePCR fragment can then be used to isolate a full length cDNA clone by avariety of methods. For example, the amplified fragment can be labeledand used to screen a cDNA library, such as a bacteriophage cDNA library.Alternatively, the labeled fragment can be used to isolate genomicclones via the screening of a genomic library.

PCR technology can also be used to isolate full length cDNA sequences.For example, RNA can be isolated, following standard procedures, from anappropriate cellular or tissue source (i.e., one known, or suspected, toexpress a NHP gene). A reverse transcription (RT) reaction can beperformed on the RNA using an oligonucleotide primer specific for themost 5′ end of the amplified fragment for the priming of first strandsynthesis. The resulting RNA/DNA hybrid may then be “tailed” using astandard terminal transferase reaction, the hybrid may be digested withRNase H, and second strand synthesis may then be primed with acomplementary primer. Thus, cDNA sequences upstream of the amplifiedfragment can be isolated. For a review of cloning strategies that can beused, see e.g., Sambrook et al., 1989, supra.

A cDNA encoding a mutant NHP sequence can be isolated, for example, byusing PCR. In this case, the first cDNA strand may be synthesized byhybridizing an oligo-dT oligonucleotide to MRNA isolated from tissueknown or suspected to be expressed in an individual putatively carryinga mutant NHP allele, and by extending the new strand with reversetranscriptase. The second strand of the cDNA is then synthesized usingan oligonucleotide that hybridizes specifically to the 5′ end of thenormal sequence. Using these two primers, the product is then amplifiedvia PCR, optionally cloned into a suitable vector, and subjected to DNAsequence analysis through methods well known to those of skill in theart. By comparing the DNA sequence of the mutant NHP allele to that of acorresponding normal NHP allele, the mutation(s) responsible for theloss or alteration of function of the mutant NHP gene product can beascertained.

Alternatively, a genomic library can be constructed using DNA obtainedfrom an individual suspected of or known to carry a mutant NHP allele(e.g., a person manifesting a NHP-associated phenotype such as, forexample, immune disorders, obesity, high blood pressure, etc.), or acDNA library can be constructed using RNA from a tissue known, orsuspected, to express a mutant NHP allele. A normal NHP gene, or anysuitable fragment thereof, can then be labeled and used as a probe toidentify the corresponding mutant NHP allele in such libraries. Clonescontaining mutant NHP sequences can then be purified and subjected tosequence analysis according to methods well known to those skilled inthe art.

Additionally, an expression library can be constructed utilizing cDNAsynthesized from, for example, RNA isolated from a tissue known, orsuspected, to express a mutant NHP allele in an individual suspected ofor known to carry such a mutant allele. In this manner, gene productsmade by the putatively mutant tissue may be expressed and screened usingstandard antibody screening techniques in conjunction with antibodiesraised against a normal NHP product, as described below. (For screeningtechniques, see, for example, Harlow, E. and Lane, eds., 1988,“Antibodies: A Laboratory Manual”, Cold Spring Harbor Press, Cold SpringHarbor.)

Additionally, screening can be accomplished by screening with labeledNHP fusion proteins, such as, for example, alkaline phosphatase-NHP orNHP-alkaline phosphatase fusion proteins. In cases where a NHP mutationresults in an expression product with altered function (e.g., as aresult of a missense or a frameshift mutation), polyclonal antibodies toNHP are likely to cross-react with a corresponding mutant NHP expressionproduct. Library clones detected via their reaction with such labeledantibodies can be purified and subjected to sequence analysis accordingto methods well known in the art.

An additional application of the described novel human polynucleotidesequences is their use in the molecular mutagenesis/evolution ofproteins that are at least partially encoded by the described novelsequences using, for example, polynucleotide shuffling or relatedmethodologies. Such approaches are described in U.S. Pat. Nos.5,830,721, 5,837,458, 6,117,679, and 5,723,323, which are hereinincorporated by reference in their entirety.

The invention also encompasses (a) DNA vectors that contain any of theforegoing NHP coding sequences and/or their complements (i.e.,antisense); (b) DNA expression vectors that contain any of the foregoingNHP coding sequences operatively associated with a regulatory elementthat directs the expression of the coding sequences (for example,baculovirus as described in U.S. Pat. No. 5,869,336 herein incorporatedby reference); (c) genetically engineered host cells that contain any ofthe foregoing NHP coding sequences operatively associated with aregulatory element that directs the expression of the coding sequencesin the host cell; and (d) genetically engineered host cells that expressan endogenous NHP sequence under the control of an exogenouslyintroduced regulatory element (i.e., gene activation). As used herein,regulatory elements include, but are not limited to, inducible andnon-inducible promoters, enhancers, operators and other elements knownto those skilled in the art that drive and regulate expression. Suchregulatory elements include but are not limited to the cytomegalovirus(hCMV) immediate early gene, regulatable, viral elements (particularlyretroviral LTR promoters), the early or late promoters of SV40adenovirus, the lac system, the trp system, the TAC system, the TRCsystem, the major operator and promoter regions of phage lambda, thecontrol regions of fd coat protein, the promoter for 3-phosphoglyceratekinase (PGK), the promoters of acid phosphatase, and the promoters ofthe yeast α-mating factors.

Where, as in the present instance, some of the described NHP peptides orpolypeptides are thought to be cytoplasmic or nuclear proteins (althoughprocessed forms or fragments can be secreted or membrane associated),expression systems can be engineered that produce soluble derivatives ofa NHP (corresponding to a NHP extracellular and/or intracellulardomains, or truncated polypeptides lacking one or more hydrophobicdomains) and/or NHP fusion protein products (especially NHP-Ig fusionproteins, i.e., fusions of a NHP domain to an IgFc), NHP antibodies, andanti-idiotypic antibodies (including Fab fragments) that can be used intherapeutic applications. Preferably, the above expression systems areengineered to allow the desired peptide or polypeptide to be recoveredfrom the culture media.

The present invention also encompasses antibodies and anti-idiotypicantibodies (including Fab fragments), antagonists and agonists of a NHP,as well as compounds or nucleotide constructs that inhibit expression ofa NHP sequence (transcription factor inhibitors, antisense and ribozymemolecules, or open reading frame sequence or regulatory sequencereplacement constructs), or promote the expression of a NHP (e.g.,expression constructs in which NHP coding sequences are operativelyassociated with expression control elements such as promoters,promoter/enhancers, etc.).

The NHPs or NHP peptides, NHP fusion proteins, NHP nucleotide sequences,antibodies, antagonists and agonists can be useful for the detection ofmutant NHPs or inappropriately expressed NHPs for the diagnosis ofdisease. The NHP proteins or peptides, NHP fusion proteins, NHPnucleotide sequences, host cell expression systems, antibodies,antagonists, agonists and genetically engineered cells and animals canbe used for screening for drugs (or high throughput screening ofcombinatorial libraries) effective in the treatment of the symptomaticor phenotypic manifestations of perturbing the normal function of a NHPin the body. The use of engineered host cells and/or animals can offeran advantage in that such systems allow not only for the identificationof compounds that bind to the endogenous receptor/ligand of a NHP, butcan also identify compounds that trigger NHP-mediated activities orpathways.

Finally, the NHP products can be used as therapeutics. For example,soluble derivatives such as NHP peptides/domains corresponding to NHPs,NHP fusion protein products (especially NHP-Ig fusion proteins, i.e.,fusions of a NHP, or a domain of a NHP, to an IgFc), NHP antibodies andanti-idiotypic antibodies (including Fab fragments), antagonists oragonists (including compounds that modulate or act on downstream targetsin a NHP-mediated pathway) can be used to directly treat diseases ordisorders. For instance, the administration of an effective amount ofsoluble NHP, or a NHP-IgFc fusion protein or an anti-idiotypic antibody(or its Fab) that mimics the NHP could activate or effectivelyantagonize the endogenous NHP or a protein interactive therewith.Nucleotide constructs encoding such NHP products can be used togenetically engineer host cells to express such products in vivo; thesegenetically engineered cells function as “bioreactors” in the bodydelivering a continuous supply of a NHP, a NHP peptide, or a NHP fusionprotein to the body. Nucleotide constructs encoding functional NHPs,mutant NHPs, as well as antisense and ribozyme molecules can also beused in “gene therapy” approaches for the modulation of NHP expression.Thus, the invention also encompasses pharmaceutical formulations andmethods for treating biological disorders.

Various aspects of the invention are described in greater detail in thesubsections below.

5.1 THE NHP SEQUENCES

The cDNA sequences and corresponding deduced amino acid sequences of thedescribed NHPs are presented in the Sequence Listing.

Expression analysis has provided evidence that the described NHPs can beexpressed in a relatively narrow range of human tissues. In addition toserine-threonine kinases, the described NHPs also share significantsimilarity to a range of additional kinase families, including kinasesassociated with signal transduction, from a variety of phyla andspecies.

An additional application of the described novel human polynucleotidesequences is their use in the molecular mutagenesis/evolution ofproteins that are at least partially encoded by the described novelsequences using, for example, polynucleotide shuffling or relatedmethodologies. Such approaches are described in U.S. Pat. Nos. 5,830,721and 5,837,458, which are herein incorporated by reference in theirentirety.

NHP gene products can also be expressed in transgenic animals. Animalsof any species, including, but not limited to, worms, mice, rats,rabbits, guinea pigs, pigs, micro-pigs, birds, goats, and non-humanprimates, e.g., baboons, monkeys, and chimpanzees may be used togenerate NHP transgenic animals.

Any technique known in the art may be used to introduce a NHP transgeneinto animals to produce the founder lines of transgenic animals. Suchtechniques include, but are not limited to pronuclear microinjection(Hoppe and Wagner, 1989, U.S. Pat. No. 4,873,191); retrovirus mediatedgene transfer into germ lines (Van der Putten et al., 1985, Proc. Natl.Acad. Sci., USA 82:6148-6152); gene targeting in embryonic stem cells(Thompson et al., 1989, Cell 56:313-321); electroporation of embryos(Lo, 1983, Mol Cell. Biol. 3:1803-1814); and sperm-mediated genetransfer (Lavitrano et al., 1989, Cell 57:717-723); etc. For a review ofsuch techniques, see Gordon, 1989, Transgenic Animals, Intl. Rev. Cytol.115:171-229, which is incorporated by reference herein in its entirety.

The present invention provides for transgenic animals that carry the NHPtransgene in all their cells, as well as animals that carry thetransgene in some, but not all their cells, i.e., mosaic animals orsomatic cell transgenic animals. The transgene may be integrated as asingle transgene or in concatamers, e.g., head-to-head tandems orhead-to-tail tandems. The transgene may also be selectively introducedinto and activated in a particular cell type by following, for example,the teaching of Lasko et al., 1992, Proc. Natl. Acad. Sci. USA89:6232-6236. The regulatory sequences required for such a cell-typespecific activation will depend upon the particular cell type ofinterest, and will be apparent to those of skill in the art.

When it is desired that a NHP transgene be integrated into thechromosomal site of the endogenous NHP gene, gene targeting ispreferred. Briefly, when such a technique is to be utilized, vectorscontaining some nucleotide sequences homologous to the endogenous NHPgene are designed for the purpose of integrating, via homologousrecombination with chromosomal sequences, into and disrupting thefunction of the nucleotide sequence of the endogenous NHP gene (i.e.,“knockout” animals).

The transgene can also be selectively introduced into a particular celltype, thus inactivating the endogenous NHP gene in only that cell type,by following, for example, the teaching of Gu et al., 1994, Science,265:103-106. The regulatory sequences required for such a cell-typespecific inactivation will depend upon the particular cell type ofinterest, and will be apparent to those of skill in the art.

Once transgenic animals have been generated, the expression of therecombinant NHP gene may be assayed utilizing standard techniques.Initial screening may be accomplished by Southern blot analysis or PCRtechniques to analyze animal tissues to assay whether integration of thetransgene has taken place. The level of mRNA expression of the transgenein the tissues of the transgenic animals may also be assessed usingtechniques that include but are not limited to Northern blot analysis oftissue samples obtained from the animal, in situ hybridization analysis,and RT-PCR. Samples of NHP gene-expressing tissue, may also be evaluatedimmunocytochemically using antibodies specific for the NHP transgeneproduct.

5.2 NHPS AND NHP POLYPEPTIDES

NHPS, NHP polypeptides, NHP peptide fragments, mutated, truncated, ordeleted forms of the NHPs, and/or NHP fusion proteins can be preparedfor a variety of uses. These uses include, but are not limited to, thegeneration of antibodies, as reagents in diagnostic assays, for theidentification of other cellular gene products related to a NHP, asreagents in assays for screening for compounds that can be used aspharmaceutical reagents useful in the therapeutic treatment of mental,biological, or medical disorders and disease. Given the similarityinformation and expression data, the described NHPs can be targeted (bydrugs, oligos, antibodies, etc.) in order to treat disease, or totherapeutically augment the efficacy of therapeutic agents.

The Sequence Listing discloses the amino acid sequences encoded by thedescribed NHP-encoding polynucleotides. The NHPs display initiatormethionines that are present in DNA sequence contexts consistent witheucaryotic translation initiation sites. The NHPs do not displayconsensus signal sequences, which indicates that they may be cytoplasmicor possibly nuclear proteins, although they may also be secreted ormembrane associated.

The NHP amino acid sequences of the invention include the amino acidsequences presented in the Sequence Listing as well as analogues andderivatives thereof. Further, corresponding NHP homologues from otherspecies are encompassed by the invention. In fact, any NHP proteinencoded by the NHP nucleotide sequences described above are within thescope of the invention, as are any novel polynucleotide sequencesencoding all or any novel portion of an amino acid sequence presented inthe Sequence Listing. The degenerate nature of the genetic code is wellknown, and, accordingly, each amino acid presented in the SequenceListing, is generically representative of the well known nucleic acid“triplet” codon, or in many cases codons, that can encode the aminoacid. As such, as contemplated herein, the amino acid sequencespresented in the Sequence Listing, when taken together with the geneticcode (see, for example, Table 4-1 at page 109 of “Molecular CellBiology”, 1986, J. Darnell et al., eds., Scientific American Books, NewYork, NY, herein incorporated by reference) are genericallyrepresentative of all the various permutations and combinations ofnucleic acid sequences that can encode such amino acid sequences.

The invention also encompasses proteins that are functionally equivalentto the NHPs encoded by the presently described nucleotide sequences asjudged by any of a number of criteria, including, but not limited to,the ability to bind and modify a NHP substrate, or the ability to effectan identical or complementary downstream pathway, or a change incellular metabolism (e.g., proteolytic activity, ion flux, tyrosinephosphorylation, etc.). Such functionally equivalent NHP proteinsinclude, but are not limited to, additions or substitutions of aminoacid residues within the amino acid sequence encoded by the NHPnucleotide sequences described above, but that result in a silentchange, thus producing a functionally equivalent expression product.Amino acid substitutions may be made on the basis of similarity inpolarity, charge, solubility, hydrophobicity, hydrophilicity, and/or theamphipathic nature of the residues involved. For example, nonpolar(hydrophobic) amino acids include alanine, leucine, isoleucine, valine,proline, phenylalanine, tryptophan, and methionine; polar neutral aminoacids include glycine, serine, threonine, cysteine, tyrosine,asparagine, and glutamine; positively charged (basic) amino acidsinclude arginine, lysine, and histidine; and negatively charged (acidic)amino acids include aspartic acid and glutamic acid.

A variety of host-expression vector systems can be used to express theNHP nucleotide sequences of the invention. Where the NHP peptide orpolypeptide can exist, or has been engineered to exist, as a soluble orsecreted molecule, the soluble NHP peptide or polypeptide can berecovered from the culture media. Such expression systems also encompassengineered host cells that express a NHP, or functional equivalent, insitu. Purification or enrichment of a NHP from such expression systemscan be accomplished using appropriate detergents and lipid micelles andmethods well known to those skilled in the art. However, such engineeredhost cells themselves may be used in situations where it is importantnot only to retain the structural and functional characteristics of theNHP, but to assess biological activity, e.g., in drug screening assays.

The expression systems that may be used for purposes of the inventioninclude but are not limited to microorganisms such as bacteria (e.g., E.coli, B. subtilis) transformed with recombinant bacteriophage DNA,plasmid DNA or cosmid DNA expression vectors containing NHP nucleotidesequences; yeast (e.g., Saccharomyces, Pichia) transformed withrecombinant yeast expression vectors containing NHP nucleotidesequences; insect cell systems infected with recombinant virusexpression vectors (e.g., baculovirus) containing NHP sequences; plantcell systems infected with recombinant virus expression vectors (e.g.,cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) ortransformed with recombinant plasmid expression vectors (e.g., Tiplasmid) containing NHP nucleotide sequences; or mammalian cell systems(e.g., COS, CHO, BHK, 293, 3T3) harboring recombinant expressionconstructs containing promoters derived from the genome of mammaliancells (e.g., metallothionein promoter) or from mammalian viruses (e.g.,the adenovirus late promoter; the vaccinia virus 7.5K promoter).

In bacterial systems, a number of expression vectors may beadvantageously selected depending upon the use intended for the NHPproduct being expressed. For example, when a large quantity of such aprotein is to be produced for the generation of pharmaceuticalcompositions of or containing NHP, or for raising antibodies to a NHP,vectors that direct the expression of high levels of fusion proteinproducts that are readily purified may be desirable. Such vectorsinclude, but are not limited, to the E. coli expression vector pUR278(Ruther et al., 1983, EMBO J. 2:1791), in which a NHP coding sequencemay be ligated individually into the vector in frame with the lacZcoding region so that a fusion protein is produced; pIN vectors (Inouye& Inouye, 1985, Nucleic Acids Res. 13:3101-3109; Van Heeke & Schuster,1989, J. Biol. Chem. 264:5503-5509); and the like. pGEX vectors may alsobe used to express foreign polypeptides as fusion proteins withglutathione S-transferase (GST). In general, such fusion proteins aresoluble and can easily be purified from lysed cells by adsorption toglutathione-agarose beads followed by elution in the presence of freeglutathione. The PGEX vectors are designed to include thrombin or factorXa protease cleavage sites so that the cloned target expression productcan be released from the GST moiety.

In an insect system, Autographa californica nuclear polyhedrosis virus(AcNPV) is used as a vector to express foreign polynucleotide sequences.The virus grows in Spodoptera frugiperda cells. A NHP coding sequencecan be cloned individually into non-essential regions (for example thepolyhedrin gene) of the virus and placed under control of an AcNPVpromoter (for example the polyhedrin promoter). Successful insertion ofNHP coding sequence will result in inactivation of the polyhedrin geneand production of non-occluded recombinant virus (i.e., virus lackingthe proteinaceous coat coded for by the polyhedrin gene). Theserecombinant viruses are then used to infect Spodoptera frugiperda cellsin which the inserted sequence is expressed (e.g., see Smith et al.,1983, J. Virol. 46: 584; Smith, U.S. Pat. No. 4,215,051).

In mammalian host cells, a number of viral-based expression systems maybe utilized. In cases where an adenovirus is used as an expressionvector, the NHP nucleotide sequence of interest may be ligated to anadenovirus transcription/translation control complex, e.g., the latepromoter and tripartite leader sequence. This chimeric sequence may thenbe inserted in the adenovirus genome by in vitro or in vivorecombination. Insertion in a non-essential region of the viral genome(e.g., region E1 or E3) will result in a recombinant virus that isviable and capable of expressing a NHP product in infected hosts (e.g.,see Logan & Shenk, 1984, Proc. Natl. Acad. Sci. USA 81:3655-3659).Specific initiation signals may also be required for efficienttranslation of inserted NHP nucleotide sequences. These signals includethe ATG initiation codon and adjacent sequences. In cases where anentire NHP gene or cDNA, including its own initiation codon and adjacentsequences, is inserted into the appropriate expression vector, noadditional translational control signals may be needed. However, incases where only a portion of a NHP coding sequence is inserted,exogenous translational control signals, including, perhaps, the ATGinitiation codon, must be provided. Furthermore, the initiation codonmust be in phase with the reading frame of the desired coding sequenceto ensure translation of the entire insert. These exogenoustranslational control signals and initiation codons can be of a varietyof origins, both natural and synthetic. The efficiency of expression maybe enhanced by the inclusion of appropriate transcription enhancerelements, transcription terminators, etc. (See Bitter et al., 1987,Methods in Enzymol. 153:516-544).

In addition, a host cell strain may be chosen that modulates theexpression of the inserted sequences, or modifies and processes theexpression product in the specific fashion desired. Such modifications(e.g., glycosylation) and processing (e.g., cleavage) of proteinproducts may be important for the function of the protein. Differenthost cells have characteristic and specific mechanisms for thepost-translational processing and modification of proteins andexpression products. Appropriate cell lines or host systems can bechosen to ensure the correct modification and processing of the foreignprotein expressed. To this end, eukaryotic host cells that possess thecellular machinery for proper processing of the primary transcript,glycosylation, and phosphorylation of the expression product may beused. Such mammalian host cells include, but are not limited to, CHO,VERO, BHK, HeLa, COS, MDCK, 293, 3T3, WI38, and in particular, humancell lines.

For long-term, high-yield production of recombinant proteins, stableexpression is preferred. For example, cell lines that stably express theNHP sequences described above can be engineered. Rather than usingexpression vectors that contain viral origins of replication, host cellscan be transformed with DNA controlled by appropriate expression controlelements (e.g., promoter, enhancer sequences, transcription terminators,polyadenylation sites, etc.), and a selectable marker. Following theintroduction of the foreign DNA, engineered cells may be allowed to growfor 1-2 days in an enriched media, and then are switched to a selectivemedia. The selectable marker in the recombinant plasmid confersresistance to the selection and allows cells to stably integrate theplasmid into their chromosomes and grow to form foci, which in turn canbe cloned and expanded into cell lines. This method may advantageouslybe used to engineer cell lines that express the NHP product. Suchengineered cell lines may be particularly useful in screening andevaluation of compounds that affect the endogenous activity of the NHPproduct.

A number of selection systems may be used, including but not limited tothe herpes simplex virus thymidine kinase (Wigler, et al., 1977, Cell11:223), hypoxanthine-guanine phosphoribosyltransferase (Szybalska &Szybalski, 1962, Proc. Natl. Acad. Sci. USA 48:2026), and adeninephosphoribosyltransferase (Lowy, et al., 1980, Cell 22:817) genes, whichcan be employed in tk⁻, hgprt⁻ or aprt⁻ cells, respectively. Also,antimetabolite resistance can be used as the basis of selection for thefollowing genes: dhfr, which confers resistance to methotrexate (Wigler,et al., 1980, Natl. Acad. Sci. USA 77:3567; O'Hare, et al., 1981, Proc.Natl. Acad. Sci. USA 78:1527); gpt, which confers resistance tomycophenolic acid (Mulligan & Berg, 1981, Proc. Natl. Acad. Sci. USA78:2072); neo, which confers resistance to the aminoglycoside G-418(Colberre-Garapin, et al., 1981, J. Mol. Biol. 150:1); and hygro, whichconfers resistance to hygromycin (Santerre, et al., 1984, Gene 30:147).

Alternatively, any fusion protein can be readily purified by utilizingan antibody specific for the fusion protein being expressed. Forexample, a system described by Janknecht et al., allows for the readypurification of non-denatured fusion proteins expressed in human celllines (Janknecht, et al., 1991, Proc. Natl. Acad. Sci. USA88:8972-8976). In this system, the sequence of interest is subclonedinto a vaccinia recombination plasmid such that the sequence's openreading frame is translationally fused to an amino-terminal tagconsisting of six histidine residues. Extracts from cells infected withrecombinant vaccinia virus are loaded onto Ni²⁺.nitriloaceticacid-agarose columns and histidine-tagged proteins are selectivelyeluted with imidazole-containing buffers.

Also encompassed by the present invention are fusion proteins thatdirect the NHP to a target organ and/or facilitate transport across themembrane into the cytosol. Conjugation of NHPs to antibody molecules ortheir Fab fragments could be used to target cells bearing a particularepitope. Attaching the appropriate signal sequence to the NHP would alsotransport the NHP to the desired location within the cell. Alternativelytargeting of NHP or its nucleic acid sequence might be achieved usingliposome or lipid complex based delivery systems. Such technologies aredescribed in “Liposomes: A Practical Approach”, New, ed., OxfordUniversity Press, New York and in U.S. Pat. Nos. 4,594,595, 5,459,127,5,948,767 and 6,110,490 and their respective disclosures, which areherein incorporated by reference in their entirety. Additionallyembodied are novel protein constructs engineered in such a way that theyfacilitate transport of the NHP to the target site or desired organ,where they cross the cell membrane and/or the nucleus where the NHP canexert its functional activity. This goal may be achieved by coupling ofthe NHP to a cytokine or other ligand that provides targetingspecificity, and/or to a protein transducing domain (see generally U.S.applications Ser. No. 60/111,701 and 60/056,713, both of which areherein incorporated by reference, for examples of such transducingsequences) to facilitate passage across cellular membranes and canoptionally be engineered to include nuclear localization.

5.3 ANTIBODIES TO NHP PRODUCTS

Antibodies that specifically recognize one or more epitopes of a NHP, orepitopes of conserved variants of a NHP, or peptide fragments of a NHPare also encompassed by the invention. Such antibodies include but arenot limited to polyclonal antibodies, monoclonal antibodies (mAbs),humanized or chimeric antibodies, single chain antibodies, Fabfragments, F(ab′)₂ fragments, fragments produced by a Fab expressionlibrary, anti-idiotypic (anti-Id) antibodies, and epitope-bindingfragments of any of the above.

The antibodies of the invention can be used, for example, in thedetection of NHP in a biological sample and may, therefore, be utilizedas part of a diagnostic or prognostic technique whereby patients may betested for abnormal amounts of NHP. Such antibodies may also be utilizedin conjunction with, for example, compound screening schemes for theevaluation of the effect of test compounds on expression and/or activityof a NHP expression product. Additionally, such antibodies can be usedin conjunction gene therapy to, for example, evaluate the normal and/orengineered NHP-expressing cells prior to their introduction into thepatient. Such antibodies may additionally be used as a method for theinhibition of abnormal NHP activity. Thus, such antibodies may,therefore, be utilized as part of treatment methods.

For the production of antibodies, various host animals may be immunizedby injection with the NHP, a NHP peptide (e.g., one corresponding to afunctional domain of a NHP), truncated NHP polypeptides (NHP in whichone or more domains have been deleted), functional equivalents of theNHP or mutated variant of the NHP. Such host animals may include but arenot limited to pigs, rabbits, mice, goats, and rats, to name but a few.Various adjuvants may be used to increase the immunological response,depending on the host species, including but not limited to Freund'sadjuvant (complete and incomplete), mineral salts such as aluminumhydroxide or aluminum phosphate, chitosan, surface active substancessuch as lysolecithin, pluronic polyols, polyanions, peptides, oilemulsions, and potentially useful human adjuvants such as BCG (bacilleCalmette-Guerin) and Corynebacterium parvum. Alternatively, the immuneresponse could be enhanced by combination and or coupling with moleculessuch as keyhole limpet hemocyanin, tetanus toxoid, diphtheria toxoid,ovalbumin, cholera toxin or fragments thereof. Polyclonal antibodies areheterogeneous populations of antibody molecules derived from the sera ofthe immunized animals.

Monoclonal antibodies, which are homogeneous populations of antibodiesto a particular antigen, can be obtained by any technique that providesfor the production of antibody molecules by continuous cell lines inculture. These include, but are not limited to, the hybridoma techniqueof Kohler and Milstein, (1975, Nature 256:495-497; and U.S. Pat. No.4,376,110), the human B-cell hybridoma technique (Kosbor et al., 1983,Immunology Today 4:72; Cole et al., 1983, Proc. Natl. Acad. Sci. USA80:2026-2030), and the EBV-hybridoma technique (Cole et al., 1985,Monoclonal Antibodies And Cancer Therapy, Alan R. Liss, Inc., pp.77-96). Such antibodies may be of any immunoglobulin class includingIgG, IgM, IgE, IgA, IgD and any subclass thereof. The hybridomaproducing the mAb of this invention may be cultivated in vitro or invivo. Production of high titers of mAbs in vivo makes this the presentlypreferred method of production.

In addition, techniques developed for the production of “chimericantibodies” (Morrison et al., 1984, Proc. Natl. Acad. Sci.,81:6851-6855; Neuberger et al., 1984, Nature, 312:604-608; Takeda etal., 1985, Nature, 314:452-454) by splicing the genes from a mouseantibody molecule of appropriate antigen specificity together with genesfrom a human antibody molecule of appropriate biological activity can beused. A chimeric antibody is a molecule in which different portions arederived from different animal species, such as those having a variableregion derived from a murine mAb and a human immunoglobulin constantregion. Such technologies are described in U.S. Pat. Nos. 6,075,181 and5,877,397 and their respective disclosures, which are hereinincorporated by reference in their entirety. Also encompassed by thepresent invention is the use of fully humanized monoclonal antibodies asdescribed in U.S. Pat. No. 6,150,584 and respective disclosures, whichare herein incorporated by reference in their entirety.

Alternatively, techniques described for the production of single chainantibodies (U.S. Pat. No. 4,946,778; Bird, 1988, Science 242:423-426;Huston et al., 1988, Proc. Natl. Acad. Sci. USA 85:5879-5883; and Wardet al., 1989, Nature 341:544-546) can be adapted to produce single chainantibodies against NHP expression products. Single chain antibodies areformed by linking the heavy and light chain fragments of the Fv regionvia an amino acid bridge, resulting in a single chain polypeptide.

Antibody fragments that recognize specific epitopes may be generated byknown techniques. For example, such fragments include, but are notlimited to: the F(ab′)₂ fragments, which can be produced by pepsindigestion of the antibody molecule and the Fab fragments, which can begenerated by reducing the disulfide bridges of the F(ab′)₂ fragments.Alternatively, Fab expression libraries may be constructed (Huse et al.,1989, Science, 246:1275-1281) to allow rapid and easy identification ofmonoclonal Fab fragments with the desired specificity.

Antibodies to a NHP can, in turn, be utilized to generate anti-idiotypeantibodies that “mimic” a given NHP, using techniques well known tothose skilled in the art. (See, e.g., Greenspan & Bona, 1993, FASEB J7(5):437-444; and Nissinoff, 1991, J. Immunol. 147(8):2429-2438). Forexample antibodies that bind to a NHP domain and competitively inhibitthe binding of NHP to its cognate receptor/ligand can be used togenerate anti-idiotypes that “mimic” the NHP and, therefore, bind,activate, or neutralize a NHP, NHP receptor, or NHP ligand. Suchanti-idiotypic antibodies or Fab fragments of such anti-idiotypes can beused in therapeutic regimens involving a NHP mediated pathway.

Additionally given the high degree of relatedness of mammalian NHPs, thepresently described knock-out mice (having never seen NHP, and thusnever been tolerized to NHP) have a unique utility, as they can beadvantageously applied to the generation of antibodies against thedisclosed mammalian NHP (i.e., NHP will be immunogenic in NHP knock-outanimals).

The present invention is not to be limited in scope by the specificembodiments described herein, which are intended as single illustrationsof individual aspects of the invention, and functionally equivalentmethods and components are within the scope of the invention. Indeed,various modifications of the invention, in addition to those shown anddescribed herein will become apparent to those skilled in the art fromthe foregoing description. Such modifications are intended to fallwithin the scope of the appended claims. All cited publications,patents, and patent applications are herein incorporated by reference intheir entirety.

4 1 2301 DNA homo sapiens 1 atggccagca ccaggagtat cgagctggag cactttgaggaacgggacaa aaggccgcgg 60 ccggggtcgc ggagaggggc ccccagctcc tccgggggcagcagcagctc gggccccaag 120 gggaacgggc tcatccccag tccggcgcac agtgcccactgcagcttcta ccgcacgcgg 180 accctgcagg ccctcagctc ggagaagaag gccaagaaggcgcgcttcta ccggaacggg 240 gaccgctact tcaagggcct ggtgtttgcc atctccagcgaccgcttccg gtcyttcgat 300 gcgctcctca tagagctcac ccgctccctg tcggacaacgtgaacctgcc ccagggtgtc 360 cgcactatct acaccatcga cggcagccgg aaggtcaccagcctggacga gctgctggaa 420 ggtgagagtt acgtgtgtgc atccaatgaa ccatttcgtaaagtcgatta caccaaaaat 480 attaatccaa actggtctgt gaacatcaag ggtgggacatcccgagcgct ggctgctgcc 540 tcctctgtga aaagtgaagt aaaagaaagt aaagatttcatcaaacccaa gttagtgact 600 gtgattcgaa gtggagtgaa gcctagaaaa gccgtgcggatccttctgaa taaaaagact 660 gctcattcct ttgaacaagt cttaacagat atcaccgaagccattaaact agactcagga 720 gtcgtcaaga ggctctgcac cctggatgga aagcaggttacttgtctgca agactttttt 780 ggtgatgacg atgtttttat tgcatgtgga ccagaaaaatttcgttatgc ccaagatgac 840 tttgtcctgg atcatagtga atgtcgtgtc ctgaagtcatcttattctcg atcctcagct 900 gttaagtatt ctggatccaa aagccctggg ccctctcgacgcagcaaatc accagcttca 960 gttaatggaa ctcccagcag ccaactttct actcctaaatctacgaaatc ctccagttcc 1020 tctccaacta gtccaggaag tttcagagga ttaaagcagatttctgctca tggcagatct 1080 tcttccaatg taaacggtgg acctgagctt gaccgttgcataagtcctga aggtgtgaat 1140 ggaaacagat gctctgaatc atcaactctt cttgagaaatacaaaattgg aaaggtcatt 1200 ggtgatggca attttgcagt agtcaaagag tgtatagacaggtccactgg aaaggagttt 1260 gccctaaaga ttatagacaa agccaaatgt tgtggaaaggaacacctgat tgagaatgaa 1320 gtgtcaatac tgcgccgagt gaaacatccc aatatcattatgctggtcga ggagatggaa 1380 acagcaactg agctctttct ggtgatggaa ttggtcaaaggtggagatct ctttgatgca 1440 attacttcgt cgaccaagta cactgagaga gatggcagtgccatggtgta caacttagcc 1500 aatgccctca ggtatctcca tggcctcagc atcgtgcacagagacatcaa accagagaat 1560 ctcttggtgt gtgaatatcc tgatggaacc aagtctttgaaactgggaga ctttgggctt 1620 gcgactgtgg tagaaggccc tttatacaca gtctgtggcacacccactta tgtggctcca 1680 gaaatcattg ctgaaactgg ctatggcctg aaggtggacatttgggcagc tggtgtgatc 1740 acatacatac ttctctgtgg attcccacca ttccgaagtgagaacaatct ccaggaagat 1800 ctcttcgacc agatcttggc tgggaagctg gagtttccggccccctactg ggataacatc 1860 acggactctg ccaaggaatt aatcagtcaa atgcttcaggtaaatgttga agctcggtgt 1920 accgcgggac aaatcctgag tcacccctgg gtgtcagatgatgcctccca ggagaataac 1980 atgcaagctg aggtgacagg taaactaaaa cagcactttaataatgcgct ccccaaacag 2040 aacagcacta ccaccggggt ctccgtcatc atgaacacggctctagataa ggaggggcag 2100 attttctgca gcaagcactg tcaagacagc ggcaggcctgggatggagcc catctctcca 2160 gttcctccct cagtggagga gatccctgtg cctggggaagcagtcccggc ccccacccct 2220 ccggaatctc ccacccccca ctgtcctccc gctgccccgggtggtgagcg ggcaggaacc 2280 tggcgccgcc accgagactg a 2301 2 766 PRT homosapiens 2 Met Ala Ser Thr Arg Ser Ile Glu Leu Glu His Phe Glu Glu ArgAsp 1 5 10 15 Lys Arg Pro Arg Pro Gly Ser Arg Arg Gly Ala Pro Ser SerSer Gly 20 25 30 Gly Ser Ser Ser Ser Gly Pro Lys Gly Asn Gly Leu Ile ProSer Pro 35 40 45 Ala His Ser Ala His Cys Ser Phe Tyr Arg Thr Arg Thr LeuGln Ala 50 55 60 Leu Ser Ser Glu Lys Lys Ala Lys Lys Ala Arg Phe Tyr ArgAsn Gly 65 70 75 80 Asp Arg Tyr Phe Lys Gly Leu Val Phe Ala Ile Ser SerAsp Arg Phe 85 90 95 Arg Ser Phe Asp Ala Leu Leu Ile Glu Leu Thr Arg SerLeu Ser Asp 100 105 110 Asn Val Asn Leu Pro Gln Gly Val Arg Thr Ile TyrThr Ile Asp Gly 115 120 125 Ser Arg Lys Val Thr Ser Leu Asp Glu Leu LeuGlu Gly Glu Ser Tyr 130 135 140 Val Cys Ala Ser Asn Glu Pro Phe Arg LysVal Asp Tyr Thr Lys Asn 145 150 155 160 Ile Asn Pro Asn Trp Ser Val AsnIle Lys Gly Gly Thr Ser Arg Ala 165 170 175 Leu Ala Ala Ala Ser Ser ValLys Ser Glu Val Lys Glu Ser Lys Asp 180 185 190 Phe Ile Lys Pro Lys LeuVal Thr Val Ile Arg Ser Gly Val Lys Pro 195 200 205 Arg Lys Ala Val ArgIle Leu Leu Asn Lys Lys Thr Ala His Ser Phe 210 215 220 Glu Gln Val LeuThr Asp Ile Thr Glu Ala Ile Lys Leu Asp Ser Gly 225 230 235 240 Val ValLys Arg Leu Cys Thr Leu Asp Gly Lys Gln Val Thr Cys Leu 245 250 255 GlnAsp Phe Phe Gly Asp Asp Asp Val Phe Ile Ala Cys Gly Pro Glu 260 265 270Lys Phe Arg Tyr Ala Gln Asp Asp Phe Val Leu Asp His Ser Glu Cys 275 280285 Arg Val Leu Lys Ser Ser Tyr Ser Arg Ser Ser Ala Val Lys Tyr Ser 290295 300 Gly Ser Lys Ser Pro Gly Pro Ser Arg Arg Ser Lys Ser Pro Ala Ser305 310 315 320 Val Asn Gly Thr Pro Ser Ser Gln Leu Ser Thr Pro Lys SerThr Lys 325 330 335 Ser Ser Ser Ser Ser Pro Thr Ser Pro Gly Ser Phe ArgGly Leu Lys 340 345 350 Gln Ile Ser Ala His Gly Arg Ser Ser Ser Asn ValAsn Gly Gly Pro 355 360 365 Glu Leu Asp Arg Cys Ile Ser Pro Glu Gly ValAsn Gly Asn Arg Cys 370 375 380 Ser Glu Ser Ser Thr Leu Leu Glu Lys TyrLys Ile Gly Lys Val Ile 385 390 395 400 Gly Asp Gly Asn Phe Ala Val ValLys Glu Cys Ile Asp Arg Ser Thr 405 410 415 Gly Lys Glu Phe Ala Leu LysIle Ile Asp Lys Ala Lys Cys Cys Gly 420 425 430 Lys Glu His Leu Ile GluAsn Glu Val Ser Ile Leu Arg Arg Val Lys 435 440 445 His Pro Asn Ile IleMet Leu Val Glu Glu Met Glu Thr Ala Thr Glu 450 455 460 Leu Phe Leu ValMet Glu Leu Val Lys Gly Gly Asp Leu Phe Asp Ala 465 470 475 480 Ile ThrSer Ser Thr Lys Tyr Thr Glu Arg Asp Gly Ser Ala Met Val 485 490 495 TyrAsn Leu Ala Asn Ala Leu Arg Tyr Leu His Gly Leu Ser Ile Val 500 505 510His Arg Asp Ile Lys Pro Glu Asn Leu Leu Val Cys Glu Tyr Pro Asp 515 520525 Gly Thr Lys Ser Leu Lys Leu Gly Asp Phe Gly Leu Ala Thr Val Val 530535 540 Glu Gly Pro Leu Tyr Thr Val Cys Gly Thr Pro Thr Tyr Val Ala Pro545 550 555 560 Glu Ile Ile Ala Glu Thr Gly Tyr Gly Leu Lys Val Asp IleTrp Ala 565 570 575 Ala Gly Val Ile Thr Tyr Ile Leu Leu Cys Gly Phe ProPro Phe Arg 580 585 590 Ser Glu Asn Asn Leu Gln Glu Asp Leu Phe Asp GlnIle Leu Ala Gly 595 600 605 Lys Leu Glu Phe Pro Ala Pro Tyr Trp Asp AsnIle Thr Asp Ser Ala 610 615 620 Lys Glu Leu Ile Ser Gln Met Leu Gln ValAsn Val Glu Ala Arg Cys 625 630 635 640 Thr Ala Gly Gln Ile Leu Ser HisPro Trp Val Ser Asp Asp Ala Ser 645 650 655 Gln Glu Asn Asn Met Gln AlaGlu Val Thr Gly Lys Leu Lys Gln His 660 665 670 Phe Asn Asn Ala Leu ProLys Gln Asn Ser Thr Thr Thr Gly Val Ser 675 680 685 Val Ile Met Asn ThrAla Leu Asp Lys Glu Gly Gln Ile Phe Cys Ser 690 695 700 Lys His Cys GlnAsp Ser Gly Arg Pro Gly Met Glu Pro Ile Ser Pro 705 710 715 720 Val ProPro Ser Val Glu Glu Ile Pro Val Pro Gly Glu Ala Val Pro 725 730 735 AlaPro Thr Pro Pro Glu Ser Pro Thr Pro His Cys Pro Pro Ala Ala 740 745 750Pro Gly Gly Glu Arg Ala Gly Thr Trp Arg Arg His Arg Asp 755 760 765 32298 DNA homo sapiens 3 atggccagca ccaggagtat cgagctggag cactttgaggaacgggacaa aaggccgcgg 60 ccggggtcgc ggagaggggc ccccagctcc tccgggggcagcagcagctc gggccccaag 120 gggaacgggc tcatccccag tccggcgcac agtgcccactgcagcttcta ccgcacgcgg 180 accctgcagg ccctcagctc ggagaagaag gccaagaaggcgcgcttcta ccggaacggg 240 gaccgctact tcaagggcct ggtgtttgcc atctccagcgaccgcttccg gtcyttcgat 300 gcgctcctca tagagctcac ccgctccctg tcggacaacgtgaacctgcc ccagggtgtc 360 cgcactatct acaccatcga cggcagccgg aaggtcaccagcctggacga gctgctggaa 420 ggtgagagtt acgtgtgtgc atccaatgaa ccatttcgtaaagtcgatta caccaaaaat 480 attaatccaa actggtctgt gaacatcaag ggtgggacatcccgagcgct ggctgctgcc 540 tcctctgtga aaagtgaagt aaaagaaagt aaagatttcatcaaacccaa gttagtgact 600 gtgattcgaa gtggagtgaa gcctagaaaa gccgtgcggatccttctgaa taaaaagact 660 gctcattcct ttgaacaagt cttaacagat atcaccgaagccattaaact agactcagga 720 gtcgtcaaga ggctctgcac cctggatgga aagcaggttacttgtctgca agactttttt 780 ggtgatgacg atgtttttat tgcatgtgga ccagaaaaatttcgttatgc ccaagatgac 840 tttgtcctgg atcatagtga atgtcgtgtc ctgaagtcatcttattctcg atcctcagct 900 gttaagtatt ctggatccaa aagccctggg ccctctcgacgcagcaaatc accagcttca 960 gttaatggaa ctcccagcag ccaactttct actcctaaatctacgaaatc ctccagttcc 1020 tctccaacta gtccaggaag tttcagagga ttaaagatttctgctcatgg cagatcttct 1080 tccaatgtaa acggtggacc tgagcttgac cgttgcataagtcctgaagg tgtgaatgga 1140 aacagatgct ctgaatcatc aactcttctt gagaaatacaaaattggaaa ggtcattggt 1200 gatggcaatt ttgcagtagt caaagagtgt atagacaggtccactggaaa ggagtttgcc 1260 ctaaagatta tagacaaagc caaatgttgt ggaaaggaacacctgattga gaatgaagtg 1320 tcaatactgc gccgagtgaa acatcccaat atcattatgctggtcgagga gatggaaaca 1380 gcaactgagc tctttctggt gatggaattg gtcaaaggtggagatctctt tgatgcaatt 1440 acttcgtcga ccaagtacac tgagagagat ggcagtgccatggtgtacaa cttagccaat 1500 gccctcaggt atctccatgg cctcagcatc gtgcacagagacatcaaacc agagaatctc 1560 ttggtgtgtg aatatcctga tggaaccaag tctttgaaactgggagactt tgggcttgcg 1620 actgtggtag aaggcccttt atacacagtc tgtggcacacccacttatgt ggctccagaa 1680 atcattgctg aaactggcta tggcctgaag gtggacatttgggcagctgg tgtgatcaca 1740 tacatacttc tctgtggatt cccaccattc cgaagtgagaacaatctcca ggaagatctc 1800 ttcgaccaga tcttggctgg gaagctggag tttccggccccctactggga taacatcacg 1860 gactctgcca aggaattaat cagtcaaatg cttcaggtaaatgttgaagc tcggtgtacc 1920 gcgggacaaa tcctgagtca cccctgggtg tcagatgatgcctcccagga gaataacatg 1980 caagctgagg tgacaggtaa actaaaacag cactttaataatgcgctccc caaacagaac 2040 agcactacca ccggggtctc cgtcatcatg aacacggctctagataagga ggggcagatt 2100 ttctgcagca agcactgtca agacagcggc aggcctgggatggagcccat ctctccagtt 2160 cctccctcag tggaggagat ccctgtgcct ggggaagcagtcccggcccc cacccctccg 2220 gaatctccca ccccccactg tcctcccgct gccccgggtggtgagcgggc aggaacctgg 2280 cgccgccacc gagactga 2298 4 765 PRT homosapiens 4 Met Ala Ser Thr Arg Ser Ile Glu Leu Glu His Phe Glu Glu ArgAsp 1 5 10 15 Lys Arg Pro Arg Pro Gly Ser Arg Arg Gly Ala Pro Ser SerSer Gly 20 25 30 Gly Ser Ser Ser Ser Gly Pro Lys Gly Asn Gly Leu Ile ProSer Pro 35 40 45 Ala His Ser Ala His Cys Ser Phe Tyr Arg Thr Arg Thr LeuGln Ala 50 55 60 Leu Ser Ser Glu Lys Lys Ala Lys Lys Ala Arg Phe Tyr ArgAsn Gly 65 70 75 80 Asp Arg Tyr Phe Lys Gly Leu Val Phe Ala Ile Ser SerAsp Arg Phe 85 90 95 Arg Ser Phe Asp Ala Leu Leu Ile Glu Leu Thr Arg SerLeu Ser Asp 100 105 110 Asn Val Asn Leu Pro Gln Gly Val Arg Thr Ile TyrThr Ile Asp Gly 115 120 125 Ser Arg Lys Val Thr Ser Leu Asp Glu Leu LeuGlu Gly Glu Ser Tyr 130 135 140 Val Cys Ala Ser Asn Glu Pro Phe Arg LysVal Asp Tyr Thr Lys Asn 145 150 155 160 Ile Asn Pro Asn Trp Ser Val AsnIle Lys Gly Gly Thr Ser Arg Ala 165 170 175 Leu Ala Ala Ala Ser Ser ValLys Ser Glu Val Lys Glu Ser Lys Asp 180 185 190 Phe Ile Lys Pro Lys LeuVal Thr Val Ile Arg Ser Gly Val Lys Pro 195 200 205 Arg Lys Ala Val ArgIle Leu Leu Asn Lys Lys Thr Ala His Ser Phe 210 215 220 Glu Gln Val LeuThr Asp Ile Thr Glu Ala Ile Lys Leu Asp Ser Gly 225 230 235 240 Val ValLys Arg Leu Cys Thr Leu Asp Gly Lys Gln Val Thr Cys Leu 245 250 255 GlnAsp Phe Phe Gly Asp Asp Asp Val Phe Ile Ala Cys Gly Pro Glu 260 265 270Lys Phe Arg Tyr Ala Gln Asp Asp Phe Val Leu Asp His Ser Glu Cys 275 280285 Arg Val Leu Lys Ser Ser Tyr Ser Arg Ser Ser Ala Val Lys Tyr Ser 290295 300 Gly Ser Lys Ser Pro Gly Pro Ser Arg Arg Ser Lys Ser Pro Ala Ser305 310 315 320 Val Asn Gly Thr Pro Ser Ser Gln Leu Ser Thr Pro Lys SerThr Lys 325 330 335 Ser Ser Ser Ser Ser Pro Thr Ser Pro Gly Ser Phe ArgGly Leu Lys 340 345 350 Ile Ser Ala His Gly Arg Ser Ser Ser Asn Val AsnGly Gly Pro Glu 355 360 365 Leu Asp Arg Cys Ile Ser Pro Glu Gly Val AsnGly Asn Arg Cys Ser 370 375 380 Glu Ser Ser Thr Leu Leu Glu Lys Tyr LysIle Gly Lys Val Ile Gly 385 390 395 400 Asp Gly Asn Phe Ala Val Val LysGlu Cys Ile Asp Arg Ser Thr Gly 405 410 415 Lys Glu Phe Ala Leu Lys IleIle Asp Lys Ala Lys Cys Cys Gly Lys 420 425 430 Glu His Leu Ile Glu AsnGlu Val Ser Ile Leu Arg Arg Val Lys His 435 440 445 Pro Asn Ile Ile MetLeu Val Glu Glu Met Glu Thr Ala Thr Glu Leu 450 455 460 Phe Leu Val MetGlu Leu Val Lys Gly Gly Asp Leu Phe Asp Ala Ile 465 470 475 480 Thr SerSer Thr Lys Tyr Thr Glu Arg Asp Gly Ser Ala Met Val Tyr 485 490 495 AsnLeu Ala Asn Ala Leu Arg Tyr Leu His Gly Leu Ser Ile Val His 500 505 510Arg Asp Ile Lys Pro Glu Asn Leu Leu Val Cys Glu Tyr Pro Asp Gly 515 520525 Thr Lys Ser Leu Lys Leu Gly Asp Phe Gly Leu Ala Thr Val Val Glu 530535 540 Gly Pro Leu Tyr Thr Val Cys Gly Thr Pro Thr Tyr Val Ala Pro Glu545 550 555 560 Ile Ile Ala Glu Thr Gly Tyr Gly Leu Lys Val Asp Ile TrpAla Ala 565 570 575 Gly Val Ile Thr Tyr Ile Leu Leu Cys Gly Phe Pro ProPhe Arg Ser 580 585 590 Glu Asn Asn Leu Gln Glu Asp Leu Phe Asp Gln IleLeu Ala Gly Lys 595 600 605 Leu Glu Phe Pro Ala Pro Tyr Trp Asp Asn IleThr Asp Ser Ala Lys 610 615 620 Glu Leu Ile Ser Gln Met Leu Gln Val AsnVal Glu Ala Arg Cys Thr 625 630 635 640 Ala Gly Gln Ile Leu Ser His ProTrp Val Ser Asp Asp Ala Ser Gln 645 650 655 Glu Asn Asn Met Gln Ala GluVal Thr Gly Lys Leu Lys Gln His Phe 660 665 670 Asn Asn Ala Leu Pro LysGln Asn Ser Thr Thr Thr Gly Val Ser Val 675 680 685 Ile Met Asn Thr AlaLeu Asp Lys Glu Gly Gln Ile Phe Cys Ser Lys 690 695 700 His Cys Gln AspSer Gly Arg Pro Gly Met Glu Pro Ile Ser Pro Val 705 710 715 720 Pro ProSer Val Glu Glu Ile Pro Val Pro Gly Glu Ala Val Pro Ala 725 730 735 ProThr Pro Pro Glu Ser Pro Thr Pro His Cys Pro Pro Ala Ala Pro 740 745 750Gly Gly Glu Arg Ala Gly Thr Trp Arg Arg His Arg Asp 755 760 765

What is claimed is:
 1. An isolated nucleic acid molecule comprising anucleotide sequence drawn from the group consisting of SEQ ID NO:1 andSEQ ID NO:3.
 2. An isolated nucleic acid molecule comprising anucleotide sequence that: (a) encodes the amino acid sequence shown inSEQ ID NO:2; and (b) hybridizes under stringent conditions to thenucleotide sequence of SEQ ID NO:1 or the complement thereof.
 3. Anisolated nucleic acid molecule comprising a nucleotide sequence encodingthe amino acid sequence shown in SEQ ID NO:2.
 4. An isolated nucleicacid molecule comprising a nucleotide sequence encoding the amino acidsequence shown in SEQ ID NO:4.